Mice

Wild-type and IL-6 KO (B6.129S2-Il6(tm1Kopf/J)) mice with a C57BL/6 genetic background were purchased from Jackson Laboratory (Bar Harbor, Maine), bred, and kept in pathogen-free animal facilities in accordance with FDA Center for Veterinary Medicine guidelines. Neonatal (5- to 7-day-old) and adult (6- to 10-week-old) mice were used for immunization experiments. All animal procedures were approved by FDA’s Institutional Animal Care and Use Committee (Protocol 2017-48). For euthanasia, adult and neonatal mice were exposed to CO2 inhalation. CO2 was introduced at a rate of at least 30% chamber volume per minute. CO2 inhalation was followed by cervical dislocation for adult mice and decapitation for neonatal mice. Anesthesia was induced by administering isoflurane, set at 3–4% for 1–2 min in the induction box and the flow rate of O2 is set at 1.0 L/min.

Immunization

Tetanus toxoid conjugated type 14 pneumococcal polysaccharide (PPS14-TT) vaccine was manufactured as described42. PPS14-TT vaccine was emulsified with aluminum hydroxide [Al(OH)3] (Thermo Fischer, Waltham, MA). Aluminum hydroxide constituted 1/4th of injection volume. For IL-6 co-injection experiments, PPS14-TT together with recombinant IL-6 (500 ng/adult and 100 ng/neonate (R&D systems, Minneapolis, MN)) was emulsified with aluminum hydroxide. The adjuvant CpG 1826 (TCCATGACGTTCCTGACGTT) was synthesized at FDA core facility. The CpG (10 μg per neonate mouse) containing PPS14-TT vaccine was emulsified with aluminum hydroxide by stirring for 30 min prior to injection. One and 0.5 μg of vaccines were injected in 150 μl and 30 μl volumes i.p. per adult and neonatal mice, respectively.

Antibody for FACS analysis

Single-cell suspensions were prepared from splenocytes. Dead cells were stained by incubating cell suspensions with Zombie Aqua (BioLegend, Cat # 423102) diluted at 1:1000 dilution in PBS for 15 min at room temperature. Cells were washed and stained using FACS buffer containing 2% FBS, 0.5 M EDTA in PBS. The following antibodies were used for surface staining at room temperature for 30 min: α-CD4 (BioLegend, 1:100 dilution, clone GK1.55, Cat # 100434), α-B220 (BioLegend, 1:100 dilution, clone RA3-6B2, Cat # 103244), α-PD-1 (BD Biosciences, 1:100 dilution, clone J43 Cat # 566831 or BioLegend, 1:100 dilution, clone 29F.1A12, Cat # 135206), α-CXCR5 biotin (BD Biosciences, 1:100 dilution, clone 2G8, Cat # 551960), α-GL7 (BioLegend, 1:100 dilution, clone GL-7, Cat # 144612), α-CD95 (BD Biosciences, 1:100 dilution, clone J02, Cat # 563647), α-IL-2Rα (BioLegend, 1:100 dilution, clone PC61, Cat # 102026), α-IL-2Rβ (BioLegend, 1:100 dilution, clone TM-β1, 123214), α-CD19 (BioLegend, 1:100 dilution, clone 6D5, Cat # 115523), α-CD3 (BioLegend, 1:100 dilution, clone 17A2, Cat # 100204), α-CD11c (BioLegend, 1:100 dilution, clone N418, Cat # 117336). To detect biotinylated CXCR5, cells were further incubated with streptavidin-BV-421 (BD Biosciences, 1:100 dilution, Cat # 563259) for 30 min at room temperature. For intracellular staining, samples were fixed with the FoxP3 Fix/Perm buffer set, following the manufacturer’s (ThermoFisher Scientific, eBioscience, Waltham, MA) instructions. Samples were then intracellularly stained with α-FoxP3 (BD Biosciences, 1:100 dilution, clone MF23, Cat # 560401 or BioLegend, 1:100 dilution, clone 150D, Cat # 320012), α-IL-2 (BD Biosciences, 1:100 dilution, clone JES6-5H4, Cat # 554429) or α-IL-6 (BioLegend, 1:100 dilution, clone MP5-20F3, Cat # 504508) antibodies for 30 min at room temperature. Flow cytometry data were acquired on Fortessa or Fortessa X20 flow cytometers (BD Biosciences) and analyzed using the FlowJo software v10.8.1 (FlowJo, Ashland, OR).

In vitro stimulation of cells

For the assessment of IL-2 production, single-cell suspensions of splenocytes were stimulated with or without PMA (25 ng/ml, Sigma-Aldrich) and ionomycin (500 ng/ml, Invitrogen) in the presence of Brefeldin A (1:1000, Invitrogen) at 37 °C for 4 h. After incubation, dead cells were stained with Zombie Aqua for 15 min at room temperature followed by surface marker staining. The cells were then fixed and permeabilized with FoxP3 Fix/Perm buffer set (ThermoFisher) and incubated with antibodies for α-IL-2 (BD Biosciences, 1:100, JES6-5H4) for 30 min at room temperature and analyzed in flow cytometry.

Phospho proteins (p-STAT5 and p-STAT3) FACS Analysis

For p-STAT5 measurement, single-cell suspensions were incubated in culture media supplemented with 10% FBS, alone or with mouse recombinant IL-2 (R&D Systems, 50 ng/ml) for 15 min at 37 °C. After washing with FACS buffer containing 1% FBS with 1 mM EDTA, cells were first fixed with BD Cytofix fixation buffer (BD Biosciences) for 10 min at 37 °C, and then permeabilized with pre-chilled BD Phosphoflow buffer III (BD Biosciences) for 10 min at 4 °C. Cell surface antibodies and p-STAT5 (BD Biosciences, 1:100 dilution, pY694, clone 47, Cat # 612598) antibody were incubated together in FACS buffer for 30 min at room temperature. To detect biotinylated CXCR5, cells were further incubated with streptavidin-BV421 (BD Biosciences, 1:100 dilution) for 30 min at room temperature. For detection of TFR cells, cells were washed and FoxP3 antibody was added to the permeabilization buffer (eBioscience) for 30 min at room temperature. For p-STAT3 staining, the cells were fixed and permeabilized as described for p-STAT5. Cell surface antibodies and p-STAT3 (BioLegend, 1:100 dilution, pY705, clone 13A3-1, Cat # 651006) antibody were incubated together in FACS buffer for 30 min at room temperature followed by CXCR5 and FoxP3 staining.

Measurement of antibody titers against PPS14

Serum antibody levels were measured in ELISA 4 weeks post immunization. For antibody measurement, 96-well plates were coated with purified PPS14 (ATCC, Manassas, Virginia) at 10 μg/ml in PBS (pH of 7) for 2 h at room temperature and then blocked for 1 h at room temperature with 5 % neonatal calf serum (Millipore Sigma, St. Louis, MO) in PBS. Serum samples (1:20 dilution) were serially diluted and 100 μl of diluted samples were transferred on coated plates for overnight incubation. After washing, wells were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG-Fc or IgA antibody (Bethyl Laboratories, Waltham, MA) for 3 h at room temperature. For detection, 100 μl of KPL SureBlue TMB microwell peroxidase substrate (Seracare, Gaithersburg, MD) is added to the wells and incubated for 15–30 min followed by addition of stop solution (KPL TMB BlueSTOP solution, Seracare). The absorbance is measured at 450 nm.

Statistical analysis

All statistical analyses were performed using Prism 9 (GraphPad). Unpaired student’s t-test and One-way ANOVA was used for all comparisons; data represented as mean ± SEM are shown. P values < 0.05 were considered statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. The data points in the graphs represent measurements taken from distinct samples in one experiment.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.